Immunoassays exploiting the properties of an optical technique known as total internal reflection (abbreviated “TIR”) are proving to be a valuable tool for detection of analytes at concentrations of 10−10 to 10−13 molar or below, without a wash step. When a light beam traveling in a waveguide is totally internally reflected at the interface between the waveguide and an adjacent medium having a lower refractive index, a portion of the electromagnetic field of the TIR light penetrates shallowly into the adjacent medium. This phenomenon is termed an “evanescent penetration” or “evanescent light”. The intensity of evanescent light drops off exponentially with distance from the waveguide surface.
Binding assays, in general, are based on the strong affinity of a selected “capture” molecule to specifically bind a desired analyte. The capture molecule/analyte pair can be an antibody/antigen pair or its converse, a receptor/ligand pair or its converse, etc., as known in the art. In a fluorescent binding assay, the binding of the analyte to the antibody is monitored by a tracer molecule which emits fluorescent light in response to excitation by an input light beam.
One of several possible schemes for exploiting the properties of evanescent light fields for fluorescence measurements is as follows. If an antibody is immobilized on an optical structure in which a light beam is being propagated by TIR, the resulting evanescent light can be used to selectively excite tracer molecules that are bound (whether directly or indirectly) to the immobilized antibody. Tracer molecules free in solution beyond the evanescent penetration depth are not excited and, therefore, do not emit fluorescence. For silica-based optical materials or optical plastics such as polystyrene, with the adjacent medium being an aqueous solution, the evanescent penetration depth is generally about 1000 to 2000 Å (angstroms). The amount of fluorescence is thus a measure of the amount of tracer bound to the immobilized capture molecules. The amount of bound tracer in turn depends on the amount of analyte present, in a manner determined by the specifics of the immunoassay procedure.
U.S. Pat. Nos. RE 33,064 to Carter, 5,081,012 to Flanagan et al., 4,880,752 to Keck, 5,166,515 to Attridge, and 5,156,976 to Slovacek and Love, and EP publication Nos. 0 517 516 and 0 519 623, both by Slovacek et al., all disclose apparatus for immunoassays utilizing evanescent sensing principles.
Desirably, an immunosensor should be capable of accurately and repeatably detecting analyte molecules at concentrations of 10−13 M (molar) to 10−15 M and preferably below. At present, such sensitivity is not believed to be available in a commercially practical and affordable immunosensor. Also desirably, an immunosensor should provide multiple “channels”, that is, the capacity for measuring multiple analytes and multiple measurements of the same analyte, on the same waveguide substrate. Such an immunosensor would allow both self-calibration with known standards and screening for a panel of different analytes selected for a particular differential diagnostic procedure.
One approach to improving the sensitivity (lowering the detection limits) of fluorescent immunosensors, proposed by Ives et al. (Ives, J. T.; Reichert, W. M.; Lin, J. N.; Hlady, V.; Reinecke, D.; Suci, P. A.; Van Wagenen, R. A.; Newby, K.; Herron, J.; Dryden, P. and Andrade, J. D. “Total Internal Reflection Fluorescence Surface Sensors” in A. N. Chester, S. Martellucci and A. M. Verga Scheggi Eds., Optical Fiber Sensors, NATO ASI Series E, Vol. 132, 391-397, 1987), is to use waveguides which are very thin, perhaps about 1 mm in thickness. Such thin waveguides may provide higher evanescent intensity and a reflection density of 500-1000 reflections/cm or more. However, the potential lowering of the detection limit by use of thin-film waveguides is achievable only if the waveguide material is nonfluorescent and low-loss. Most present evanescent immunosensing technology (“thick” waveguides) utilizes silica glass (SiO2), which is intrinsically nonfluorescent. Only the purest grades of silica, for example, UV grade quartz which is rather expensive, lack the additives and impurities that fluoresce (Dierker et al., 1987).
Further, one cannot simply fabricate silica-on-silica waveguides by depositing SiO2 onto a quartz substrate because there would be no refractive index difference. Instead, one must either (1) fabricate a glass waveguide of higher refractive index than the underlying silica substrate, or (2) deposit a silica waveguide onto a transparent substrate of a lower refractive index. Therefore, other materials must be employed.
Thin film waveguides have been described by Sloper et al. (“A planar indium phosphate monomode waveguide evanescent field immunosensor,” Sensors and Actuators, B1: 589-591, 1990) and Zhou et al. (“An evanescent fluorescence biosensor using ion-exchanged buried waveguides and the enhancement of peak fluorescence”, Biosensors and Bioelectronics, 6:595-607, 1991. However, neither of these devices was capable of achieving detection of analyte concentrations significantly below 10−10 molar. The waveguide structure of Sloper et al. was of the gradient-index type, formed by diffusion of a dopant into the silica base, which results in a drop-off of dopant concentration with distance from the interface. The waveguide of Zhou et al. had only a single “channel” (measurement region).
Therefore, a need exists for an optical structure useful in an evanescent sensing immunoassay which provides increased levels of propagated TIR light and increased evanescent field intensity, as well as multiple measurement regions. Such an optical structure should desirably be capable of detection of analyte concentrations of 10−13 M and preferably below 10−15 M. A need also remains for an immunosensor including such an optical structure which is sufficiently inexpensive and practical to produce as a commercial device, and which provides accurate and repeatable results in the hands of nonskilled persons. Still further, a need also remains for a biosensor capable of detecting ions, as opposed to hormones or other biological molecules.